scholarly journals Sequence of formation of molecular forms of plasminogen and plasmin-inhibitor complexes in plasma activated by urokinase or tissue-type plasminogen activator

1984 ◽  
Vol 223 (1) ◽  
pp. 179-187 ◽  
Author(s):  
S Thorsen ◽  
S Müllertz ◽  
E Suenson ◽  
P Kok
Keyword(s):  
Plasminogen Activator ◽  
Effector Function ◽  
Tissue Type ◽  
Molecular Forms ◽  
Plasminogen Activation ◽  
Tissue Type Plasminogen Activator ◽  
Fibrin Formation ◽  
Type Plasminogen Activator ◽  
Plasmin Inhibitor ◽  
Stimulation Of

The pathway of plasminogen transformation was studied in plasma, particularly in relation to fibrin formation and the subsequent stimulation of plasminogen activation. Plasminogen was activated by urokinase (low fibrin-affinity) or tissue-type plasminogen activator (high fibrin-affinity). Formation of 125I-labelled free and inhibitor-bound plasminogen derivatives was quantified after their separation by acetic acid/urea/polyacrylamide-gel electrophoresis. In plasma activator converted Glu-plasminogen (residues 1-790) into Glu-plasmin, which was complexed to alpha 2-plasmin inhibitor. When this inhibitor was saturated, Glu-plasmin was autocatalytically converted into Lys-plasmin (residues 77-790). No plasmin-catalysed Lys-plasminogen formation was observed. Upon fibrin formation, activation initially followed the same Glu-plasminogen-into-Glu-plasmin conversion pathway, and stimulation of plasminogen activation was only observed with tissue-type plasminogen activator. In agreement with the emergence of novel effector function, on early plasmin cleavage of fibrin [Suenson, Lützen & Thorsen (1984) Eur. J. Biochem. 140, 513-522] the fibrin-binding of Glu-plasminogen increased when solid-phase fibrin showed evident signs of degradation. This was associated with the formation of considerable amounts of the more easily activatable Lys-plasminogen, most of which was fibrin-bound. At the same time the rate of plasmin formation with urokinase increased over that in unclotted plasma and the rate of plasmin formation with tissue-type plasminogen activator accelerated. Altogether these processes favoured enhanced fibrin degradation. The rates of Lys-plasminogen and plasmin formation abruptly decreased after lysis of fibrin, probably owing to a compromised effector function on further fibrin degradation.

A Simple, Sensitive Spectrophotometric Assay for Extrinsic (Tissue-Type) Plasminogen Activator Applicable to Measurements in Plasma

1982 ◽  
Vol 48 (03) ◽  
pp. 266-269 ◽  
Author(s):  
J H Verheijen ◽  
E Mullaart ◽  
G T G Chang ◽  
C Kluft ◽  
G Wijngaards
Keyword(s):  
Plasminogen Activator ◽  
Cyanogen Bromide ◽  
Spectrophotometric Assay ◽  
Tissue Type ◽  
Plasminogen Activation ◽  
Tissue Type Plasminogen Activator ◽  
Type Plasminogen Activator ◽  
Specific Stimulation ◽  
Assay Conditions ◽  
Stimulation Of

SummaryAn indirect spectrophotometric assay for extrinsic plasminogen activator has been devised, which is based on the parabolic assay of Drapier et al. (5). The system contains activator, plasminogen, the synthetic plasmin substrate H-D-Val-Leu-Lys-pNA (S-2251, Kabi) and a mixture of soluble fibrinogen fragments prepared by treatment of fibrinogen with cyanogen bromide. The addition of these fibrinogen fragments considerably enhances the sensitivity and specificity of the method owing to specific stimulation of the plasminogen activation by extrinsic plasminogen activator.The assay conditions were optimized and the application for extrinsic plasminogen activator measurements in plasma euglobulin fractions is demonstrated.

SITES IN TISSUE-TYPE PLASMINOGEN ACTIVATOR INVOLVED IN THE INTERACTION WITH FIBRIN, PLASMINOGEN AND LOW MOLECULAR WEIGHT LIGANDS

1987 ◽  
Author(s):  
J M Verheijen ◽  
M P M Caspers ◽  
G A W de Munk ◽  
B E Enger-Valk ◽  
G T G Chang ◽  
...  
Keyword(s):  
Binding Site ◽  
Plasminogen Activator ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Tissue Type ◽  
Plasminogen Activation ◽  
Tissue Type Plasminogen Activator ◽  
Type Plasminogen Activator ◽  
Arginine Residues ◽  
Stimulation Of

Tissue-type plasminogen activator (t-PA) activates the proenzyme plasminogen to the active protease plasmin which degrades fibrin. The unique properties of t-PA, fibrin binding and stimulation of activity by fibrin make it an interesting molecule for specific thrombolysis. t-PA is thought to consist of five structural regions designated finger (F), growth factor (G), kringle 1 (Kl), kringle 2 (K2) and protease (P). Previous studies have shown that the interaction of t-PA with fibrin is mediated by the F and K2 regions.Mutated t-PA cDNA molecules were expressed in Chinese hamster ovary cells and t-PA analog proteins were purified from serum free culture media using affinity chromatography with immobilized monoclonal antibodies. Besides FGK1K2P (native t-PA) the following analogs were used GK1K2P, klK2P, K2P, P, FP and FGKlk2P (kl and k2 have partial deletions of the kringle). All the molecules comprising K2P could be stimulated in plasminogen activation activity by fibrinogen fragments comparable to normal t-PA. The activities of FP and FGKlk2P were only slightly influenced by these fragments. It was shown that the fibrin binding site in K2 was plasminogen dependent whereas that in F was not. K2 was found to contain a binding site for lysine, 6-amino-hexanoic acid but also 6-amino-hexane and thus to differ from the high affinity lysine binding sites in plasminogen.Chemical modification of lysine and arginine residues in t-PA with citraconic anhydride and cyclohexanedione respectively, revealed no involvement of these residues in interaction with lysine or analogs nor in stimulation of activity by fibrinogen fragments. Arginine modification led to inhibition of plasminogen activation activity, both in the presence and absence of fibrinogen fragments, but the amidolytic activity as measured with a tripeptide paranitroanilide was not changed. The involvement of one or more arginine residues in interaction of t-PA with plasminogen seems likely.

Assay of Functional Plasminogen in Rat Plasma Applicable to Experimental Studies of Thrombolysis

2000 ◽  
Vol 84 (08) ◽  
pp. 299-306 ◽  
Author(s):  
Kristian Bangert ◽  
Sixtus Thorsen
Keyword(s):  
Plasminogen Activator ◽  
Experimental Studies ◽  
Fold Increase ◽  
Rat Plasma ◽  
Tissue Type ◽  
Plasminogen Activation ◽  
Type Plasminogen Activator ◽  
Plasmin Inhibitor

SummaryAn improved sensitive, specific, precise and accurate assay of plasminogen in rat plasma was developed. It is performed in 96-well microtiter plates and can be completed within one hour. The assay is based on activation of plasminogen by human urokinase-type plasminogen activator (uPA) and simultaneous measurement of generated plasmin with the specific plasmin substrate H-D-Val-Phe-Lys-4-nitroanilide (S-2390), using purified native rat plasminogen for calibration. The concentration of S-2390 in the final reaction mixture during the whole reaction period is much greater than the K m value (≈20 µM) for rat plasmin-cleavage of S-2390 ensuring that hydrolysis of substrate follows zero order kinetics and that the substrate produces a 20-35 fold decrease in rate of inhibition of plasmin by its target inhibitors in plasma. Analogous to the human system the target plasma inhibitors of rat plasmin are shown to be plasmin inhibitor and α-macroglobulins. Tranexamic acid (0.8 mM) is incorporated in the reaction mixture resulting in a 19-fold increase in the rate of plasminogen activation and presumably an about 50-fold decrease in the rate of inhibition of generated plasmin by plasmin inhibitor. The assay is suitable for accurate measurement of plasminogen in samples obtained from animals containing pharmacological concentrations of uPA or tissue-type plasminogen activator (tPA) in their plasma when in vitro plasminogen activation is blocked at pH 5 by collecting blood in acidic anticoagulant. Judged from in vitro experiments formation of catalytic active plasmin-α-macroglobulin complexes during massive activation of plasminogen in vivo does not interfere with the assay.

scholarly journals Localization in the fibrinogen γ-chain of a new site that is involved in the acceleration of the tissue-type plasminogen activator-catalysed activation of plasminogen

1992 ◽  
Vol 283 (1) ◽  
pp. 187-191 ◽  
Author(s):  
O Yonekawa ◽  
M Voskuilen ◽  
W Nieuwenhuizen
Keyword(s):  
Plasminogen Activator ◽  
Sequence Data ◽  
Disulphide Bond ◽  
Void Volume ◽  
Tissue Type ◽  
Plasminogen Activation ◽  
Main Band ◽  
Tissue Type Plasminogen Activator ◽  
Type Plasminogen Activator ◽  
Water Ph

In previous publications [e.g. Voskuilen, Vermond, Veeneman, Van Boom, Klasen, Zegers & Nieuwenhuizen (1987) J. Biol. Chem. 262, 5944-5946] we have shown that fibrin(ogen) chain fragment A alpha-(148-160) contains a site that contributes to the acceleration of Glu-plasminogen activation by tissue-type plasminogen activator (t-PA). In contrast with fibrin, this peptide, however, does not enhance the rate of mini-plasminogen activation. Therefore, possibly more stimulatory sites than A alpha-(148-160) are present in fibrin. In the present investigation we have localized a possible second type of stimulatory site in the fibrin(ogen) molecule. A whole CNBr digest of fibrinogen was applied to a Bio-Gel P-2 column run in water, pH 4. Two peaks with stimulatory activity were observed, one at the void volume and one between the void volume and the total volume. The former contained the previously described stimulating fragment FCB-2 [which comprises A alpha-(148-160)]; the latter had not been observed before and was characterized further. The stimulating material in the low-M(r) fraction of the Bio-Gel P-2 column was precipitated at pH 8.3 in a virtually pure form. It has a high tryptophan content, and an M(r) of 6500 as assessed by SDS/PAGE. On reduction, a main band of M(r) 2500 is seen, plus a weakly staining band of M(r) 4000. These properties plus the amino acid sequence data identify the fragment as FCB-5. FCB-5 consists of two chains, i.e. gamma-(311-336) and gamma-(337-379), linked by a single disulphide bond between Cys-gamma-326 and Cys-gamma-339. Both these chains and the disulphide bond appear to be essential for rate enhancement. FCB-5 enhances the activation rates of Glu-, mini- and micro-plasminogen, with all five kringles, only kringle V and without kringles respectively. FCB-5 binds t-PA, but none of the plasminogen forms binds to FCB-5. This indicates that the rate enhancements induced by FCB-5 are due to an effect on t-PA.

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